Title

Author

Date of Award

5-1990

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Sartorelli, Alan C.

Abstract

Modulation of surface transferrin receptor (TfR) activity has been associated with leukemia cell differentiation and proliferation. To examine the mechanisms involved in regulating this event, surface receptor protein and mRNA levels were measured in HL-60 promyelocytic leukemia cells induced to differentiate along the myelocytic and monocytic pathways. Surface TfR down-regulation which occurs during granulocytic differentiation by dimethylsulfoxide, retinoic acid, or aclacinomycin A appears to be kinetically compatible with reduced biosynthesis resulting from reductions in the level of steady-state mRNA. In contrast, biosynthetic modulation does not appear to mediate the intial surface receptor down-regulation seen during 12-O-tetradecanoylphorbol-13-acetate-induced macrophagic differentiation. However, a reduction in levels of receptor message appears to contribute to the maintenance of diminished surface TfR activity in these 12-O-tetradecanoylphorbol-13-acetate-treated cells. The down-regulation of surface TfR, in contrast, is not an early event during monocytic differentiation by 1alpha,25-dihydroxyvitamin D3. A common feature of myelocytic and monocytic differentiating cells is the complete inhibition of cellular proliferation observed within 10 to 16 hours following a four-fold reduction in surface TfR. We conclude that the early decline in surface TfR levels precludes its regulation as a consequence of the decrease in proliferation, but rather implicates its role in the programmed cessation of growth which is requisite for the terminal differentiation of these cells.A series of phosphodiester and phosphorothioate antisense oligonucleotides were synthesized against the human TfR. The phosphorothioate analogues exhibited marked biological efficacy in culture, as assessed by inhibition of surface TfR content and HL-60 growth, whereas their unmodified phosphodiester counterparts were ineffective. Phosphorothioate oligonucleotides were more resistant to hydrolysis by serum and cellular nucleases, and were more readily taken up by cells than the phosphodiesters. A length effect was observed, with antisense 30-mers exhibiting greater TfR inhibitory activity than 17-mers. The degree of surface receptor inhibition observed, however, was not strictly sequence dependent. This suggested that the photphorothioate oligonucleotides may be biologically active in eukaryotic cells by processes other than classical antisense complementary binding to mRNA. (Abstract shortened with permission of author.)